Project Summary Traumatic brain injuries (TBIs) are a major societal and public health concern with over 2.8 million TBIs reported each year in the US. Mild TBIs (mTBIs), accounting for over 80% of TBIs, can be difficult to diagnose. Because no FDA-approved therapeutic intervention for mTBI exists, a period of rest to allow symptom resolution is the primary treatment approach. It is imperative that while symptomatic, a person recovering from mTBI avoid sustaining a second mTBI, as multiple mTBIs greatly increases the risk for prolonged disability. The period of brain vulnerability after mTBI, however, extends beyond the resolution of clinical symptoms, underscoring the vital need for accurate assessment of neurophysiological recovery in order to mitigate the risks associated with repeated head injury. Unlike moderate and severe TBI, which are typically associated with neuron death and vascular disruption, mTBI results in more subtle physiological and cellular changes, such as metabolic distress and alterations in cerebral blood flow (CBF). We hypothesize that mTBI induces acute, transient changes in CBF that, coupled with metabolic dysregulation, form the basis of the window of vulnerability to repeated mTBI. We predict, therefore, that a second injury induced during the period of acute CBF alteration will result in worsened outcome as reflected by greater perturbations in CBF and metabolism. Our group has developed a novel multiple-wavelength speckle contrast diffuse correlation tomography (MW- scDCT) technique that yields non-invasive, longitudinal, regional mapping of CBF and oxygenation in mice. We have validated our technique against established methods and demonstrated its utility in detecting CBF changes in rodents. In Aim 1, we will first use MW-scDCT to measure cortical and hippocampal CBF and oxygenation after single closed head injury (CHI) to monitor temporal changes and determine the time to recovery. We will then determine whether normalization of CBF is required to prevent synergistic effects of a second CHI on cerebral hemodynamics. Neurovascular coupling and cerebrovascular reactivity will be assessed at selected time points to inform potential mechanisms underlying CBF changes. Finally, quantitative analysis of cerebrovascular structure and communication will be performed to identify anatomical plasticity or damage. In Aim 2, a targeted metabolomics approach will be used to identify metabolite profiles in cortical tissue and plasma which are unique to mice with single or repeated mTBI. We will further test whether restoration of the metabolome coincides with normalization of CBF after mTBI. Such a finding would support a dual-pronged approach for assessing concussion recovery through noninvasive CBF monitoring and assessment of plasma metabolite biomarkers. These studies will pair metabolomics with our innovative MW- scDCT technique for monitoring cerebral hemodynamics to provide new insights into the neurophysiological determinants of the brain?s vulnerability to repeated mTBI and support the development of diagnostic and prognostic biomarkers for mTBI.